Integration is an essential step of the retrovirus life cycle. Our previously studies indicated that two molecular clones of human T-cell leukemia virus type I (HTLV-I), K30p and K34p vary in sequence by 18 bp yet show significant functional difference in that K30p mediates persistent virus production whereas K34p does not. Fourteen (14) full length HTLV-I chimeric clones were prepared from K30p and K34p and transfected into a rabbit T cell line; only clones with the pol and pX genes of K30p mediated stable provirus integration. Neither clone K34p nor any constructs with the K34p pX gene and pol gene [encoding substitutions in Pol (A247T) and integrase (K227Q)] caused provirus integration. In order to determine the influence of the substitution K227Q in integrase on viral integration, three HTLV-I clones containing a single nucleotide (C, G, and T respectively) substitution at position 227 were constructed and transfected into a rabbit T cell line. Intact virus particles that contained HTLV-I RNA and functional reverse transcriptase (RT) as well as HTLV-I gag protein were detected in a transient fashion but no integrated proviruses were detected in any of the three transfectants. All transfectants and their parent clone K30p show a similar distribution of viral mRNA in the nucleus vs, cytoplasm.These results demonstrate that substitution at K227 of integrase abolishes virus integration but does not affect particle production and nuclear export of unspliced viral mRNA in T cell line. Identification of HTLV-I receptor is important for HTLV-I pathogenesis, therapeutic research and vaccine. Although several candidates of the receptor for HTLV-I have previously been proposed, the identity of the HTLV-I receptor has not been determined. Cell-free virus infection is a useful method to search for receptor of retrovirus such as HIV. However, unlike HIV, the infectivity of cell-free HTLV-I is very low and in vitro infection by HTLV-I usually requires cocultivation of target cells with infected cells. We have developed a cell-free HTLV-I infection assay system, in which fresh cell-free HTLV-I successfully infected mammalian cell lines. Using this approach, several human cell lines were tested for the susceptibility to HTLV-I infection and two groups of cell lines that show different susceptibility to HTLV-I infection were identified.